There have been many attempts to create in vitro models permitting the evaluation of the effect of active agents on skin in a way that is predictive of the in vivo situation. The value of such methods is many fold. They permit elimination of live animal tests, they permit cheaper evaluation of potentially effective or toxic agents and they can permit more precise control of experimental variables than is possible using living animal or human skin. Most recent efforts to develop such systems have used cultured human epidermal cells grown on one of several possible substrates designed to mimic the dermis of skin. These systems have developed a high level of sophistication and are now available commercially, but at relatively high prices that reduce their potential areas of application. Furthermore, even the best of these models fail to reproduce the key function of skin adequately—that is they fail to generate a stratum corneum of equal impermeability to real skin. This failure to produce a good barrier is a critical failure as it means that the models cannot be used well to predict the effect of topically applied materials and, more critically, are suspect when investigation of the effect of agents on the process of stratum corneum formation are required.
An alternative to cell culture methods to create useful models is to use skin taken from animals or humans and maintained in culture. Such “organ culture” systems have the great advantage that the skin is already fully formed, with a fully functional barrier and all other cells types in their normal organization. Skin organ culture was investigated extensively as a model, especially in the 1960-80 time period. While much progress was made, the utility of the models was limited by two critical problems.
First, it proved difficult to devise culture conditions that maintained the normal structure of skin. Particular problems were the loss of tissue organization, necrosis and excessive and atypical growth of the epidermis. After much effort however conditions were devised that sustained essentially normal cellular organization—the peak of this art was represented by the work of Chapman et al ((Chapman S. et al, 1989. “A fully differentiating epidermal model with extended viability: development and partial characterization”. J. Investigative Dermatology 93, 762-768) who succeeded in maintaining pig skin in culture for up to 28 days with excellent maintenance of tissue structure provided the size of the skin sample was as small as 4 mm diameter.
This success however highlighted a second critical problem that had been found by many investigators. Good maintenance of tissue organization could only be achieved by reducing the size of the piece of skin being cultured to less than about 1 sq. cm.—in fact normally less than 6 mm in diameter.
For example                Tammi et al (“Histometric analysis of human skin in organ culture”. J. Investigative Dermatology 73, 138-140, 1979) found that pieces of skin of 6 mm diameter suffered degeneration after 5 days in culture        Henge et al (Expression of naked DNA in Human, Pig and Mouse Skin. J. Clin. Invest. 97, 2911(1996)) cultured larger pieces of skin up 10 mm by 20 mm but could only sustain them for 24 hours        Flaxman et al (“Organ culture of human skin in chemically defined medium”. J. Invest. Dermatol. 64, 96 (1975)) achieved maintenance of pieces of skin of 2 mm diameter for several days        Rijnkels et al (Photoprotection by antioxidants against UVB radiation induced damage in pig skin organ culture. Radiation Research 159, 210 (2003) and “Time and dose related ultraviolet B damage in viable pig skin explants held in a newly developed organ culture system”, Photochem Photobiol 73, 499 (2001)) succeeded in culturing large pieces of skin up to 60 mm but observed degeneration after 48 hours        
This limitation had two serious consequences. First, the behavior of the culture was affected significantly by the trauma experienced at the cut edges of the skin sample. Second, it was extremely difficult to apply test agents topically to such small pieces of skin without the agents leaking around the edges of the skin sample and thereby bypassing the skin barrier. Thus analysis of large numbers of agents in such systems was impractical.
The current inventive method seeks improvements of deficiencies in the known prior art. Among the one or more problems addressed include developing an organ culture that can sustain a large sheet of skin under conditions that maintain its normal tissue organization and response to stimuli and that permits topical application of multiple agents to the skin.